This research program represents an integrated, multidisciplinary approach aimed at gaining a deeper understanding of the central biological phenomenon of enzyme catalyzed protein glycosylation. The specific transformation under investigation involves the biosynthesis of all N- linked glycoproteins and is catalyzed by the multimeric membrane associated enzyme oligosaccharyl transferase (OT). Central to this modification is the co-translational transfer of a complex carbohydrate from a lipid-linked pyrophosphate donor to the carboxamide side-chain of an asparagine residue. The primary peptide sequence requirement for the process is very simple, and includes a minimum -Asn-Xaa-Ser/Thr- tripeptide recognition motif. This intriguing transformation involves a formal nucleophilic attack by an asparagine primary amide nitrogen, and displays remarkable selectivity considering the competing functionality within the peptidyl substrates including the "reactive" asparagine. The reaction catalyzed by OT represents the first critical step in the formation of a covalent bond between protein and carbohydrate biopolymers. This enzyme-catalyzed process is highly conserved throughout eukaryotic evolution and approximately 60% of all glycoproteins contain N-linked carbohydrates. Asparagine-linked glycosylation is critical in a number of biological functions such as intracellular targeting and intercellular recognition. Carbohydrates are also thought to be involved in a "decoy" function by masking the underlying peptide sequence from the immune system, and proteolytic or microbial attack. Additionally, for some proteins, glycosylation may play a structural role either by facilitating protein folding or by stabilizing folded motifs. The biological function of glycosylation is related to cell health; the aberrant glycosylation of proteins is associated with diseased and transformed cells. A thorough understanding of OT-mediated catalysis will also aid in the development of glycosylated eukaryotic proteins of therapeutic and diagnostic interest. The specific aims of this proposal are presented in four categories namely: l. Studies to define a complete molecular description of the OT enzyme comp x. 2. Development of peptide-based probes for the analysis of OT structure and function. 3. Synthesis and evaluation of pseudodisaccharide transition state analog inhibitors for OT. 4. Biophysical studies - Impact of glycosylation on conformation. The investigation of the origins of specificity and the generation of reactivity in enzyme-catalyzed protein glycosylation directly impact the future prospects for controlling, manipulating and emulating these key biological transformations.